Negamycin

ABSTRACT

Process for the production of an antibiotic substance called negamycin which comprises cultivating a strain of Streptomyces purpeofuscus A.T.C.C. 21470 in an aqueous carbohydrate solution containing a nitrogenous nutrient under aerobic conditions until substantial antibacterial activity is imparted to said solution and then recovering said negamycin from said solution and negamycin which is effective for inhibiting Pseudomonas, Salmonella, Shigella, Klebsiella, E.coli, Staphylococci and which has the following structure:

United States Patent Umezawa et al.

[ 51 July 25, 1972 NEGAMYCIN Inventors: Hamao Umezawa, Tokyo; Shinichi Kondo, Yokohama; Kenii Takeuchl Maeda, Tomio; Masa l-lamada, Tokyo, all of Japan Assignee: Zaidan l-lojin Biseibutsu Kagaku Kai,

Tokyo, Japan Filed: April 28, 1970 Appl. No.: 32,641

Foreign Application Priority Data Hamada et a]. J. Antibiotics 23(3) 170(1970) Primary ExaminerL0rraine A. Weinberger Assistant ExaminerJohn F. Terapane Anarne v-Brady, O'Boyle and Gates 5 7] ABSTRACT Process for the production of an antibiotic substance called negamycin which comprises cultivating a strain of Streptomyces purpeofuscus A.T.C.C. 21470 in an aqueous carbohydrate solution containing a nitrogenous nutrient under aerobic conditions until substantial antibacterial activity is imparted to said solution and then recovering said negamycin from said solution and negamycin which is effective for inhibiting Pseudomonas, Salmonella, Shigella, Klebsiella, E.c0li, Staphylococci and which has the following structure:

NH; 0 CH3 1 Claim, 2 Drawing Figures 0 0 4000 3000 2000 I800 I600 I400 I200 I000 e00 650 3500 2500 I900 I700 I500 I300 II00 900 700 PATENTEDJMS m2 SHEET 1 BF 2 0O: OOm O09 00E O09 00mm 00mm 09. com omw oom 000. com. 003 com: 009 000m 000m Q09 0 0 0m ow ow ow o ow k /|.l|\ v 922% m w k. w o q mlmm o IN VENTORS HAMAo UMEzA NA SHINICHI KoNDo KINJI MAxnA LEM/o [Anzac/u ASA AMAIJA BY ad gpzi ATTORNEYS NEGAMYCIN This invention relates to a new useful antibiotic substance called negamycin, and to its production. More particularly, it relates to processes for its production by fermentation and methods for recovery and purification. This invention embraces this antibacterial agent and its salts in dilute solutions, as crude concentrates, as crude solids and as pure solid. This substance is effective in inhibition of Pseudomonas, Salmonella, Klebsiella and Staphylococci. This substance has low toxicity and exhibits therapeutic effect on infections of Pseudomonas and other sensitive organisms in mice. This substance is useful in the cure of infections of Pseudomonas and other sensitive organisms in humans and animals.

This is now provided, according to the present invention, an antibiotic substance (and its salts) effective in inhibiting Pseudomonas, Salmonella, Klebsiella and Staphylococci, said antibiotic being soluble in water and practically insoluble in methanol, ethanol, butanol, ethyl acetate, butyl acetate, chlorofonn, and benzene, exhibiting no maximum absorption of ultraviolet light from 220-400mp., giving positive reactions to ninhydrin, red tetrazolium and Rydon-Smith and negative reactions to Sakaguchi and Molisch, exhibiting absorption bands in the infrared region of the spectrum when pelleted with potassium bromide at the following wave numbers in cm": 3,430, 3200, 3050, 2,950, 1,660, 1,590, 1,405, 1,320, 1,140 1,050, 970, 890, 820, 720, being optically active, [or] D +2.5 (c2, H O), having the molecular formula of C H N showing three pK values of 3.55, 8.10, 9.75, and having the following structure: 1

CH2-CHCHz-CHCHz-fi3-NHNCH2C 0 OH l lHg OH NH: 0 CH3 Referring to the drawings:

FIG. 1 is the infrared absorption spectrum of negamycin taken with potassium bromide, and

FIG. 2 is the nuclear magnetic resonance spectrum of negamycin in D 0.

There is provided according to the present invention the process for the production of the antibiotic negamycin which comprises cultivating a strain of S. purpeofizrcus in a nutrient medium under aerobic conditions until a substantial amount of negamycin is accumulated in said solution.

Negamycin is a new antibiotic discovered by the present inventors. The organism producing the antibiotic of the present invention was first found by the present inventors and was isolated from a soil sample collected at Myogisan, Gunma prefecture in Aug., 1964 and was given the laboratory number M890-C2. The inventors also isolated negamycin from other two strains which were given the laboratory numbers of No. MA91-M and No. MAl04-Ml. The strain No. MA9l-Ml was isolated from a soil sample collected at Nojiriko, Nagano prefecture in Jan. 1965, and the strain No. MAl04-Ml was isolated from a soil sample collected at Sakomachi, Tokushima city in Feb. 1965. These strains have many common properties with the stain No. M890-C2 and also with Streptomyces purpeofuscus. Though there were some minor differences among them, these stains were classified as Streptomyces purpeofilscus. Moreover, a type culture of S. purpeofuscus (S. purpeofilscus Yamaguchi and Saburi lSP 5283, 18? being the abbreviation of International Streptomyces Project) was confirmed by the present inventors to produce negamycin. The strain No. M890-C2 was deposited in Kogyogijutsuin Hokko Kenkyujo on May 6, 1969, and the deposite number 306 was given. It was also deposited in American Type Culture Collection as ATCC 21470.

The strain No. M890-C2 has the following characters. Under microscope, well-branched substrate mycelium develops straight to flexuous aerial mycelium without whorl branching nor spiral formation. Surface of spore is smooth.

Characters on various media (Designation of color is described in in accordance with Color Harmony Manual of Container Corporation of America):

1. On glycerol Czapeks agar medium incubated at 27 C: Pale yellow to dark yellowish brown [Spice brown 4 ni] growth forms abundant aerial mycelium of light grey color; brownish to pale reddish brown soluble pigment is produced;

2. On Krainshkys glucose asparagine agar medium incubated at 27 C.: Growth of yellowish brown [Luggage Tan 4 ne] to dark yellowish brown [Clove Brown 3 ni] produces aerial mycelium of yellowish white to light brownish gray; soluble pigment is faint brown;

3. On calcium malate agar medui incubated at 27 C.: Growth of grayish brown [Fawn 4 ig] produces scant grayish white to light gray aerial mycelium. No soluble pigment. Calcium malate around growth is solubilized strongly after 3 days incubation;

4. ln peptone solution containing 1.0 percent sodium nitrate incubated at 27 C.: Colorless to pale yellowish brown growth without aerial mycelium. No Soluble pigment or brown pigment slightly. Weak reduction of nitrate is observed;

5. On potato plug incubated at 27 C.: wrinkled growth of pale yellowish brown [Lt Gold 2 ic] to yellowish brown [Yellow Maple 3 ng], lately dark yellowish brown [Clove Brown 3 pl] to black. No aerial mycelium. Faint brownish soluble pigment;

6. On starch agar plate incubated at 27 C.: Pale yellowish brown to yellowish brown [Spice Brown 4 ni] growth produces aerial mycelium of yellowish white to light gray color. Brownish to pale reddish brown soluble pigment. Positive hydrolysis of starch;

7. On nutrient agar medium incubated at 37 C.: No growth within 14 days incubation;

8. On nutrient agar medium incubated at 27 C.: Pale reddish brown [Brick Red 6 k ng] growth produces no aerial mycelium and produces pale reddish brown soluble pigment;

9. On Loefflers coagulated serum medium incubated at 37 C.: No growth within 14 days incubation;

10. On gelatin stub cultured at 20 C.: Colorless to pale yellowish brown growth produces no aerial mycelium and produces soluble pigment of brown to dark brown color. Liquefaction of gelatin is strong; 1

11. On skimmed milk incubated at 37 C.: Colorless growth produces no aerial mycelium nor soluble pigment. After complete coagulation of milk at 4 days incubation, slow peptonization occurs;

12. On tyrosine agar medium incubated at 27 C.: Growth of colorless or gray to dark brown color produces thin aerial mycelium of white to grayish white color. Determination of tyrosinase activity is difficult because of blackish pigmentation of soluble pigment;

13. On cellulose (filter paper) incubated at 27 C.: Scant growth without decomposition of cellulose;

14. Utilization of carbohydrates on Pridham-Gottliebs basal medium incubated at 27 C.: Positive growth with starch, dextn'n, glycerol, galactose, glucose, sucrose, maltose and mannose. Negative growth with inositol, lactose, mannitol, rafiinose, rhamnose, inulin, sorbitol, fructose, xylose, arabinose, salicin and dulcitol.

The characters of the strain No. M890-C2 may be summarized as follows: It belongs to genus Streptomyces having aerial mycelium forming neither spirals nor whorls; the surface of spores is smooth; on various media it forms dark yellowish brown growth with gray or light gray to light brownish gray aerial mycelium; it forms no soluble pigment or faintly reddish brown soluble pigment very slightly; the reverse of the growth turns to black during days of the incubation; on nutrient agar at 27 C. it forms reddish brown growth and reddish brown soluble pigment; it forms melanin pigment, hydrolyzes starch and has relatively strong proteolytic activity. These properties conform to those of Streptomyces purpeofuscus Yamaguchi and Saburi described in lntemational Journal of Systematic Microbiology, 18, 364, 1968. However, the strain No. M890-C2 does not utilize lactose and xylose which are utilized by S. purpeofuscus. On the other hand, another strain No. MA104-M1 utilizes xylose. Though the strains producing negamycin isolated by the present inventors had minor differences with known species of S. purpeofiwcus, they were assigned to S. purpeofitscus. Moreover, the type culture of S. purpeofwcus (IS? 5283) was confirmed by the inventors to produce negamycin. On production of antibiotics by S. purpeofuscus Yamaguchi and Saburi there has been only simple description (J. of General and Applied Microbiology, 1, 201-234, 1955) that its culture filtrate showed inhibition against trichomonas, Gram positive bacteria and acid-fast bacteria. There was no description of production of antibiotics active against Gram negative bacteria. Therefore, though S. purpeofuscur is a known species, there has never been observation on production of negamycin until the present invention.

As it is well known, streptomyces is generally not stable and easily gives mutants and variants artificially or in nature. streptomyces purpeofuscus in the present invention includes all of these mutants and variants which produce negamycin, that is, those which can not be absolutely differentiated from S. purpeofuscus and produce negamycin.

Method of determining amount of negamycin: It is determined by an ordinary cylinder plate method using pure negamycin as the standard and the sensitive organism such as E. coli K12, Pseudomonas aeruginaosa etc.

S. purpeofiucus producing negamycin when grown under suitable conditions produces negamycin. A fermentation broth containing negamycin is prepared by inoculating spores or mycelium of the negamycin-producing organism into a suitable medium and then cultivating under aerobic condition. For the production of negamycin cultivation on a solid medium is possible, but for production of large quantities cultivation in a liquid medium is preferred. Any fermentation temperature can be employed within the range in which the negamycin-producing organism can grow and produce negamycin, although 2532 C. is preferred. Media consisting of known kinds of nutritional sources for actinomycetes are useful for production of negamycin. For example, commercial products such as pepstone, meat extract, N-Z amine, casein, soybean meal, corn steep liquor, peanuts meal, cotton sead meal, sodium nitrate, ammonium nitrate, ammonium sulfate and other nitrogenous materials such as wheat bran, rice'bran, etc. are useful as the nitrogen source. The commercially available products, such as glucose, glycerol, starch, maltose, dextrin, sucrose, lactose, soybean oils and other carbohydrates or other fats in pure or crude state are useful as the carbon source. Sodium chloride, sodium or potassium phosphate, calcium carbonate can be also added. Trace of metal salts can be added, if necessary. Any kinds of constituents which can be utilized by negamycin-producing organisms for production of negamycin is useful.

The fermentation is continued until negamycin is substantially accumulated. For example, spores and mycelia on the slant culture of S. purpeofuscus were inoculated into a medium consisting of glucose 2.0 percent, starch 2.0 percent, soybean meal 2.0 percent, yeast extract 0.5%, sodium chloride 0.25 percent, calcium carbonate 0.35 percent, CuSO -5H,O 0.0005 percent, MnCl,-7HO 0.0005 percent, ZnSOflI-LO 0.005 percent, adjusted to pH 7.0) and shake-cultered at 27 C. Then, the accumulation of negamycin was observed in 3-8 days, for instance, 20;.tg/rnl on 3rd day, 108;.tg/ml (pl-l 7.4)on 6th day, l85,u.g/ml (pH 7.6) on 7th day, l22ug/ml (pH 7.8) on 8th day.

Negamycin is relatively stable in its aqueous solution. For instance, after heating of Smg/ml negamycin solution of various pH (that is 1.8, 7.2, 9.2) at 60 C. for 30 minutes, 91 percent remained without decomposition. When 2mg/ml of negamycin solution of various pH was kept at 27 C. for a week, the following percent of negamycin remained without decomposition: 86 percent at pH 2.25, 80 percent at pH 4.90, 76 percent at pH 7.15, 89 percent at pH 9.0, and 79 percent at pH 10.2.

There is provided according to the present invention processes for extraction and purification of negamycin. Negamycin is soluble in water and exists mainly in the liquid part of the fermentation broth. Negamycin in the broth is not transferred from the broth. to organic solvents such as butanol, ethyl acetate, ether, chloroform, benzene.

With the adsorbents, negamycin can be obtained from the fermented broth or from its aqueous solution. Active carbon is one of preferred adsorbents. Negamycin adsorbed on active carbon can be eluted efliciently by aqueous methanol, aqueous butanol, aqueous acetone. It is more esily eluted, if the elution is made at acid condition with hydrochloric acid. Though negamycin has carboxylic acid group, having two basic groups the'whole molecule is basic and adsorbed by a cation exchange resin. A cation exchange resin precess is the most suitable method for extraction of negamycin from the fermented broth or its aqueous solution. All kinds of resins having carboxylic acid group or sulfonic acid group are useful and they can be used in H form, sodium form potassium form or ammonium form or the mixed forrn. Negamycin adsorbed on the cation exchange resin can be eluted with an acid such as hydrochloric acid, sulfuric acid etc, but more preferably with aqueous ammonia. Negamycin which has carboxylic acid group can be adsorbed also by an anion exchange resin. For instance, Dowex 1X2 (trade name, Dow Chemical Co., U.S.A.) in OH form absorbs negamycin and negamycin adsorbed is eluted with dilute hydrochloric acid. However, anion exchange resin having weak basic group such as Amberlite IR45 (trade name, Rhom and Haas Co., U.S.A.) in OH form can be used for neutralization of acidic solution of negamycin without substantial adsorption of this antibiotic.

Mycelium in the fermented broth can be removed by filtration or centrifugation. The femented broth including mycelium can be also directly applied to a resin column without procedure of removal of the mycelium, if a large particle which prevent the pass through the column was removed by passing through metal netz. The following is one of method suitable for isolation of negamycin from the fermented broth of S. purpeafiascus: the fermented broth is filtered or centrifuged and the filtrate or the supernatant is passes through a cation exchange resin column in 70 percent NH, form, and after the column was washed with water negamycin adsorbed was eluted with 2.0 percent NH,OH in water and the eluate containing negamycin is concentrated under reduced pressure the fractions containing negamycin is evaporated under vacuum, yielding white powder of pure negamycin; if the puri-- ty is not enough, the chromatography is repeated. On the basis of the basic property negamycin can be precipitated by addition of a water-insoluble acid, for instance, picric acid, phydroxyazobenzene-p-sulfonic acid etc. Negrnycin crystal lizes as the salt of p-'hydroxyazobenzene-p-sulfonic acid. Negamycin in these water-insoluble salts can be separated from the acids by methods know to separate a base from a water-insoluble acid, for instance, negamycin p-hydroxyazobenzene-p'-sulfonate is treated with HCl in ethanol and negamycin hydrochloride is precipitated. Water-insoluble acid such as Targitol 4, Targitol 7 (Union Carbide and Carbon Chemical Co.) and sodium laurylsulfonate can be also used for precipitation of negamycin.

Negamycin purified by methods described above is obtained as white powder. It is freely soluble in water, and insoluble in methanol, ethanol, esters, benzene. Negamycin in the aqueous solution shows end absorption, but no maximum absorption of ultrabviolet light from 220 to 400m It is amphoteric, showing strong basic property and weak acidic property. Under 3,500V and pH 1.8 bufi'er (formic acid-acetic acid-water in 25:75:900 in volume) negamycin moves by 12cm to cathode, and taking the mobility of alanine as 1.0, the mobility of negamycin is 1.4. It forms acid addition salts with hydrochloric acid, sulfuric acid, p-hydroxyazobenzene-p'-sulfonic acid, picric acid, etc. Negamycin having carboxylic acid group also forms metal salts and esters. It gives positive reactions to ninhydrin, Rydon-Srnith and negative reactions to anthrone and Sakaguchi. When heated at 105 C. in 6N HCl for 6 hours, the hydrolysate showed more than 3 ninhydrin products (the main products are three). Three main hydrolysis products are r-hydroxy-B-lysine, N,N'-dimethylhydrazine and methylamirre. Sarcosine and l-methyldhydroginovacetic acid are obtained as minor hydrolysis products.

Most purified negamycin melted gradualy from 75 C. and decomposed with foaming at 110l20 C. The following result was obtained by elemental analysis: calcd. for C l-l N O,-2H O: C 38.02, H 8.51,N 19.71, 0 33.77; found C 37.78, H 8.62, N 18.94, 0 31.70. As the titration equivalent 287 was obtained. Negamycin p-hydroxyazobenzene-p'-sulfonate is obtained as yellow-orange crystals, m.p. 180-182 C. with decomposition, which shows about one-third antibacterial activity of negamycin and which showed the following analytical result: calcd. for C,,l-l ,,N O (C,,H, N O,S) 211 0: C 47.13, H 5.27, N 13.33, 0 26.64, S 7.63; found: C 47.09, H 5.16, N 13.20, 0 25.53, S 8.01. Negamycin has three titrable pK as follows: 3.55, 8.10, 9.75. In the infrared spectrum of negamycin taken with potassium bromide, absorption bands were observed at the following wave numbers in cm: 3,430, 3,200, 3,050, 2,950, 1,660, 1,590, 1,405, 1,320, 1,140, 1,050, 970, 890, 820, 720. Negamycin p-hydroxyazobenzene-p'-sulfonate shows absorptions bands at the following waver numbers in cm": 3,550, 3400, 3,350, 3,250, 3,200, 3,050, 2,950, 1,730, 1,690, 1,600, 1,560, 1,510, 1,440, 1,430, 1,400, 1,360, 1,280-1,100, 1,060, 1,030, 1,010, 990, 960, 945, 900, 880, 850, 800, 715, 680. In this salt the band for carboxylic acid is shown at 1,730cm". The infrared spectrum of negamycin methyl ester shows the bands at the following wave numbers in cm": 3,400, 3,200, 3,000, 2,950, 1,740, 1,665, 1,600, 1,485, 1,445, 1,400, 1,220, 1,140, 1,050, 1,000, 960, 890, 720. The band for the ester group is at 1,740 and 1,220cm' The structure already shown was proposed by the present inventors for negamycin on the basis of mass spectometry of negamycin methyl ester and triacetyl negamycin methyl ester and determination of structures of hydrolysis products.

Negamycin inhibits growth of Gram negative and positive organisms. When the antibacterial effect was tested by agar streak method using meat extract-peptone agar, the following organisms were inhibited at the following concentrations (one loopful amount of 20 hours nutrient broth culture was streaked; the values in parenthesis are the concentrations of partial inhibition): Staphylococcus aureus 209p 50ug/ml; Staphylococcus aureus 193 l2.5p.glml (6.l2p.g/rnl); Staphylococcus aureus Smith SOpg/mhSarcina lutea PC1-1001 12.5[Lg/1'11l (1.56ug/m1); Bacillus subtilis NRRL-B558 25ug/m1 (12.5ug/ml); E.coli K12 3.12p.g/ml (1.56ug/ml); E.coli NIHJ 12.5;tg/ml (3.12;.tg/ml); Shigella flexneri 12.5;rg/ml; Salmonella typhosa 3.12ug/ml. Klebsiella pneumoniae PCl 602 12.5;rg/ml (6.25;.tg/ml), Seratia marscescene 12.5p.g/ml (6.25ug/ml), Proteus valgaris 0X19 6.25ug/ml; Proteus rettgeri (GN311) 12.5ug/ml (6.25pg/ml), Pseudomonas aeruginosa (No. 2) 12.5pglml; Pseudomonas aeruginosa No.3. 25p.glml (12.5;rg/ml); Pseudomonas fluorescene 6.25ug/ml (3.12pg/ml); Mycobacterium smegmatis 607 l00ug/ml. When the 20 hours nutrient broth culture was 1,000 times diluted and one loopful amount was streaked, then the growth inhibition was observed at half concentrations of those described above. In 0.5 percent peptone agar, the inhibitory effect was stronger than on the nutrient agar and the following organism were inhibited at the following concentrations (values in parenthesis are concentrations of partial inhibition): S. aureus 209p 1.56ptg/ml; S. lutea PCl 1001 5.0p.g/ml (2.5;Lg/ml); B. subtilis NRRL-B558 12.5p.glml; E.coli K12 l.56p.g/ml; E,coli NIl-U 1.56,.rg/ml; S. flexneri 3.12;.tg/ml; K.pneumonial PCI 602 6.25p.g/ml (3.12ug/ml);

SJyphosa 0.78p.g/rnl; Pr. rettgeri CN3ll l.56p.g/ml, S. mar- .rcescens 12.5ug/ml (3. l2ug/ml; Ps. aeruginosa No.3 6.25ug/ml Ps. aeruginam No. 46 3.12ug/ml (1.56;.tg/ml); M. segmatis 607 50p.g/ml. It does not inhibitc. albicans at ;.tg/m1. The inhibitory efiect of negamycin is not reduced by serum, for instance, E. coli was inhibited by 3.l2p.g/ml of negamycin in all media containing serum at 0, 5, 10, 20, 40 percent. Negamycin exhibits stronger antibacterial effect at alkaline side, for instance, the inhibitory concentrations against E. coli in 0.5 percent peptone water of various pH were as follows: pH 5.0 l2.5p.glrnl (3.12ug/ml); PH 6.0 12.5! -8/m1 (3.12ug/l); pH 7.0 3.l2p.g/ml (l.56p.glml); PH 8.0 1.56ptg/ml (0.78ug/ml); pH 9.0 1.56ug/ml (0.78ug/ml). In a cylinder plate method using 0.5 percent peptone agar and E. coli K12, 25ug/ml of negamycin showed the following inhibition diameters at the following pH: pH 6.0 16mm; pH 7.0 20mm; pH 8.0 21mm.

Negamycin has low toxicity. The intravenous injection of 400 mg/kg caused death of mice. No toxic sign was observed, when 200mg/kg was daily intraperitoneally injected to mice for 30 days. It has no delayed toxicity.

The intramuscular injection of 50mg/kg to rabbit gave high blood concentration such as 100p.g/ml at 1 hour after the administration and 80 percent was excreted into admin 24 hours indicating high concentration in urine, for instance 4,480p.g/ml in urine taken 1-2 hours after the injection. These results suggest the positive effect also in vivo. Really, negamycin was confirmed to be effective against infection of Pseudomonas aerugompsa No. 12, Klebsiella pneumoniae S-1802, Salmonella typhosa 63 and Staphylococcus aureus Smith S- 424 in mice. CD against these infections were 4.4, 5.0, 2.5 and 12.5mg/kg, respectively, when 10 MLD was infected intraperitoneally and negamycin was subcutaneously injected immediately and 6 hours after the infection.

As described above, negamycin is an antibacterial substance having low toxicity and inhibiting Gram positive and negative bacteria including Pseduomonas. This antibiotic is easily differentiated from known antibiotics by its analytical results, its infrared spectrum, its amphoteric property, its behavior on high voltage electrophoresis and its structure. Thus, negamycin is definitely a new antibiotic discovered by the present inventors.

As shown by the structure, hydrolysis of negamycin gives 1- methylhydrazino-acetic acid which was found to convert to N,N-dimethylhydrazine. l-Methyl-hydrazinoacetic acid was found by the present inventors to inhibit glutarnic acid-pyruvate transaminase and has liver toxicity. Negamycin has no such toxicity, however, considering the possible hydrolysis, negamycin should be infected for short period to treat the sensitive inventions. Negamycin is also used locally for treatment of superficial infections such as infections in skin and mucous membrane. Moreover, negamycin is useful for synthesis of negamycin analogues.

Properties of negamycin is now clear and therefore on the basis of the properties disclosed by the present invention processes for production of this antibiotic is easily deviced. The examples are shown below. However, this invention is not lirrritted to these examples. This invention includes all modifications of processes described in this invention.

EXAMPLE 1 A medium 125ml containing glucose 2 percent, starch 2 percent, soybean meal 2 percent, yeast extract 0.5 percent, NaCl 0.25 percent, CaCO 0.35%, CuSO,'5H O 0.0005%, MnCl -7l-l O 0.0005 percent, and ZnSO,'7H O 0.0005 percent was placed in a shaking flask of 500 ml. After adjusting pH to 7.0, it was sterilized at C. for 20 minutes. To this medium, 2.5m] of 2 days shaken-cultured broth of the strain No. M890-C2 was inoculated aseptically. The medium for seed culture contained glucose 1 percent, starch 1 percent, meat extract 0.75 percent, peptone 0.75 percent, NaCl 0.3 percent, MgSO '7H O 0.1percent, CuSO -5H,O 0.0007percent, FeSO,'7H,O 0.000] percent, MnCl,-7H,O 0.0008percent, and ZnSO 7H,O 0.0002percent. The shaking of 130 stroke per minute was given during the fermentation period days) and temperature was kept at 27 C. This cultured broth was centrifuged to separate mycelia and 6,000ml of the filtrate was obtained from the 60 flasks. The filtrate contained 35p.g/ml of negamycin. The filtrate was passed through a column (diameter 30 mm)containing 400 ml of Amberlite IRC-SO 70percent Na form). After washed with water, negamycin was eluted with 20percent ammonia in water. The active fraction of 600 ml was concentrated under reduced pressure, and. 144ml of the concentrated solution (pH 9.4, 800p.g/rnl) was obtained.

The concentrated solution was passed through .a column (diameter 16 mm) containing 60 ml of Dowex 1X2 (OH form). After washed with 200 ml of water, negamycin was eluted with 0.5NHC]. After neutralization with Amberlite lR-45 (OH form), the active fraction was dried under reduced pressure. The crude powder of negamycin hydrochloride of 300 mg (purity 2 lpercent) was obtained.

EXAMPLE 2 A crude powder (300 mg) of negamycin hydrochloride obtained in Example 1 was dissolved in 10 ml of water. The solution was passed through a column (diameter 16 mm) containing 60ml of Amberlite CG-50 (NH, form), followed by distilled water. The efluent was fractionated by l0 ml. The active efluent was found in fractions 57-96. From fractions 57-74, 25 mg (purity 68p.glmg) of lightly yellowish powder of negamycin was obtained. From fractions 75-85, mg of colorless powder of pure negamycin was obtained. From fractions 86-96, 19 mg (purity 9l0p.g/mg) was obtained.

EXAMPLE 3 Various kinds of ion exchange. resins were tested for isolation of negamycin. The broth filtrate of 200 ml obtained by a procedure as shown in Example 1 was passed through a column (diameter 10 mm) containing 10 ml of a resin. The column was washed with 50 ml of water, and then eluted as shown below. In all cases, no activity was recognized in efluents and water used for washing of the column. The results The medium of 10 liters containing the same constituents as shown in Example 1 was placed in a glass fermenter of 20 liters. After sterilization, 500ml of the shaken-cultured broth of the strain N0.-M890C3 was inoculated aseptically. The fermentation was operated at 27 C. for 90 hrs. under aeration and agitation. This cultured broth (pH 7.4) was centrifuged to separate mycelia and 6,460'ml (4lp.glml) of the filtrate was obtained. The filtrate was passed through a column (diameter 30 mm) containing 400 ml of Amberlite IRC-SO (70percent Na form). After washed with 3,000 ml of water, negamycin was eluted with 2percent ammonia. The active eluate of 400 ml was dried under reduced pressure, and 685mg (purity ISOug/ml) of brownish powder of crude negamycin was obtained.

3 EXAMPLE 5 Negamycin of 102mg was dissolved in 8ml of water containing l5percent ethylene glycol monomethyl ether. To this solution, 106mg of sodium p-hydroxy-azobenzene-p-sulfonate was added. The mixture was warmed to 50 C. to dissolve, and

then 0.5m] of 1N HCl was added to adjust pH to 3. After 7 EXAMPLE 6 A medium (130 liters) containing glucose 3 percent, starch 1 percent, soybean meal 2percent, dried yeast"0.5 percent, CaCO; 0.35 percent CuS0 -5H,O 0.0005 percent, MnCl,-7H ,0 0.0005 percent, and ZnSO -7H,O 0.0005 percent was placed in a 200 liters stainless steel fermenter. After sterilization at 120' C. for 30 minutes, 1.2 liters of a cultured broth of the strain No. M890-C2 were inoculated aseptically. The stirring of 200 rpm and the aeration of 25 liters air per minute were given during the fermentation period (l 13 hrs.) and'temperature was kept at 27 C. The cultured broth of 110 liters (pH 7.4, 63pflml) was filtered with the aid of 8 kg of diatomaceous earth after adjusting pH to 4.9 with 1 liter of 6N HCl. lncluding the'water used for washing of the filter, totally 1 17 liters of the filtrate were obtained. The filtrate was passed through a column containing 6.5 liters of Amberlite lRC-SO (70percent NH form). After washed with .water, negamycin was eluted with 0.5N ammonia. The active eluate of 3 liters EXAMPLE 7 The powder of 3.4 g having 29.4 percent purity obtained in Example 6 was dissolved in water of 22ml and adjusted to pH 8.8. This solution was applied to a column (diameter. 26 mm) filled with 250 ml of Amberlite CG-SO (NH, form). After washed with 500 ml of water, 0.1 percent NH OH was passed through the column. The eluate was fractionated by 20 ml. Negamycin was found .in fractions 69-8l. From fractions 69-73, 233 mg (purity percent) of pale-yellowish powder of negamycin was obtained. From fractions 74-77, 383 mg of pure negamycin was obtained. From fraction 78-81, 395 mg of pure negamycin was obtained.

Included within the scope of the present invention are negamycin and acid addition salts of negamycin with organic and inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, nitric acid, citric acid, maleic acid, malic acid, tartaric acid, benzoic acid, cinnamic acid, ascorbic acid, acetic acid, picric acid, p-hydroxyazobenzene-p-sulfonic acid, phytic acid, livopimaric-6, 8acis-endosuccinic acid, sulfamic acid, glycolic acid and mandelic acid. For terapeutic purposes use is made of salts of nontoxic acids but salts of toxic acids, e.g., p-hydroxyazobenzenev p'-sulfonic acid, are useful in isolation procedures, e.g. as

sulfapyrazine, sulfaguanidine, sulfathalidine, sulfasuxidine, sulfisoxazole, sulfamylon, phthalyl-sulfacetamide, N'-3,4- dimethylbenzoylsufanilamide, benzylsulfanil'a'mide and N-2- (Z-quinoxalyl) sulfanilamide), lipotropic agents (particularly methionine, choline, inositol and beta-sitosterol and mixtures thereof), stimulants of the central nervous system (e.g. aspirin, salicylamide, sodium gentisate, p-acetylaminophenol, phenacetin, codeine), laxatives (e.g. phenol-phtalein), sedatives (e.g. barbiturates, bromides), salts of penicillin (e.g. potassium penicillin G, procaine, penicillin G, l-ephenamine penicillin G, dibenzylamine penicillin G; these combinations are particularly useful to enable variation of the pattern of blood levels obtained), phenoxymethyl-penicillin and salts thereof, other antibiotic agents (e.g. streptomycin, dihydrostreptomycin, kanamycin, bacitracin, polymixin, tyrothricin, erythromycin, chlortetracycline, oxytetracycline, tetracycline, oleandomycin, chloramphenicol, magnamycin, novobiocin,

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,67%1742 Dated Julv 25 .1972

I fls) Hamao UMEZAWA, Shinichi KONDO, Kenji MAEDA, Tomio TAKEUCHI and Masa HAMADA It is certified that error appears in the abate-identified patent and that said Letters Patent are hereby corrected as shown below:

On the face of the patent, in the h'e ':1 5li1 1g at item [72]., v change 'the names of the inventors and cities from" Shinichi Rondo, Yokohama; Kenji Takeuc'hi Maeda',- 'Tcmic; to"'+ -Shin'ichi Rondo, Kanagawa-ken; Ke'nji Maeda, Tokyo; Tomio, Take'uchi Tokyo- At item [73] change the name 'of the Assi gne eto read asfoll lOWS: -ZAIDAN HOJIN BISEIBUTSU. KAGAKU KENKYU- KAI-r.

At item [30] in the Priority Data Statement I change "44/34827" and"44/83843 7" t0 3%827/69 &83 843 /69 respectively. 4

' Signed and sealed this l 7th day of April 1973.

(SEAL) Attest:

. ROBERT GOTTSCHALK Commissioner o f-Patents EDWARD M.'FLETCHER,JR. Attesting Officer FORM PO-1050 (10-69) uscoMM-oc boa! e-Pee O 0.8. .OVIIIIIIIIT PRINTING OIHC! "OI 0-8l0-33O 

